Team:TU Delft/16 August 2010 content
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|NEBuffer 2 | |NEBuffer 2 | ||
|✓ | |✓ | ||
- | |‘E - pAlkS - | + | |‘E - pAlkS - S’ |
|- | |- | ||
|11 | |11 | ||
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|Varies | |Varies | ||
|Yes | |Yes | ||
+ | |} | ||
+ | |||
+ | ==Ligation== | ||
+ | Following the digestion the products were [[Team:TU_Delft/protocols/ligation|ligated]] for 15 minutes at RT: | ||
+ | |||
+ | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
+ | |'''#''' | ||
+ | |'''BioBrick''' | ||
+ | |'''Fragment 1''' | ||
+ | |'''Fragment 2''' | ||
+ | |'''Destination Vector''' | ||
+ | |- | ||
+ | |1 | ||
+ | |014C | ||
+ | |‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ | ||
+ | |‘X - J61100-rubA4-J61100-rubR-B0015 - P’ | ||
+ | |‘E - pSB1C3 - P’ | ||
+ | |- | ||
+ | |2 | ||
+ | |020C | ||
+ | |‘E - J23100-X-J61100-ladA - S’ | ||
+ | |‘X - J61101-ADH - P’ | ||
+ | |‘E - pSB1C3 - P’ | ||
+ | |- | ||
+ | |3 | ||
+ | |405C | ||
+ | |‘E - J23100-X-J61101-PhPFDα - S’ | ||
+ | |‘X - J61101-PhPFDβ - P’ | ||
+ | |‘E - pSB1C3 - P’ | ||
+ | |- | ||
+ | |4 | ||
+ | |327C | ||
+ | |‘E - pCaiF - S’ | ||
+ | |‘X - B0032 - P’ | ||
+ | |‘E - pSB1C3 - P’ | ||
+ | |- | ||
+ | |5 | ||
+ | |304C | ||
+ | |‘E - pAlkS - S’ | ||
+ | |‘X - B0032 - P’ | ||
+ | |‘E - pSB1C3 - P’ | ||
+ | |- | ||
+ | |6 | ||
+ | |Ligation control | ||
+ | |None | ||
+ | |None | ||
+ | |‘E - pSB1C3 - P’ | ||
|} | |} |
Revision as of 19:19, 16 August 2010
Alkane degradation
Unfortunately last week's attempts hadn't yielded any BioBricks making use of the 2-way ligation method. We thus decided to take another stab at the 3-way ligation procedure, according to the BioBrick Assembly Manual of Ginkgo Bioworks. The BioBrick numbers we tried to construct were: 014C, 020C, 405C, 327C and 304C.
Digestion
# | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
1 | 011A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61100-alkB2-J61100-rubA3 - S’ |
2 | 013K | XbaI | PstI | HindIII | NEBuffer 2 | ✓ | ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ |
3 | 017A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61100-ladA - S’ |
4 | 018A | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - J61101-ADH - P’ |
5 | 402A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61101-PhPFDα - S’ |
6 | 403A | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - J61101-PhPFDβ - P’ |
7 | pCaiF | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - pCaiF - S’ |
8 | B0032 | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - B0032 - P’ |
9 | B0032 | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - B0032 - P’ |
10 | pAlkS | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - pAlkS - S’ |
11 | pSB1C3 | EcoRI | PstI | None | NEBuffer 2 | ✓ | ‘E - pSB1C3 - P’ |
The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:
Lane description
# | Description | Expected size (bp) | OK? |
1 | Smartladder | Varies | Yes |
2 | 011A cut | 1523, 1199, 857 | Yes |
3 | 013K cut | 1825, 1592, 1342, 43 | Yes |
4 | 017A cut | 1410, 1199, 857 | Yes |
5 | 018A cut | 1185, 872, 793, 43 | Yes |
6 | 402A cut | 1199, 857, 543 | Yes |
7 | 403A cut | 1185, 872, 397, 43 | Yes |
8 | pCaiF in pANY cut | 1277, 1143, 74 | Yes |
9 | B0032 in pSB1A2 cut | 1185, 872, 35 | Yes |
10 | B0032 in pSB1A2 cut | 1185, 872, 35 | Yes |
11 | pAlkS cut | 1272, 1138, 106 | Yes |
12 | pSB1C3 cut | 2035, 1081 | Yes (However incomplete digestion, see 3116 bp) |
15 | BioRad EZ Load Marker | Varies | Yes |
Ligation
Following the digestion the products were ligated for 15 minutes at RT:
# | BioBrick | Fragment 1 | Fragment 2 | Destination Vector |
1 | 014C | ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ | ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ | ‘E - pSB1C3 - P’ |
2 | 020C | ‘E - J23100-X-J61100-ladA - S’ | ‘X - J61101-ADH - P’ | ‘E - pSB1C3 - P’ |
3 | 405C | ‘E - J23100-X-J61101-PhPFDα - S’ | ‘X - J61101-PhPFDβ - P’ | ‘E - pSB1C3 - P’ |
4 | 327C | ‘E - pCaiF - S’ | ‘X - B0032 - P’ | ‘E - pSB1C3 - P’ |
5 | 304C | ‘E - pAlkS - S’ | ‘X - B0032 - P’ | ‘E - pSB1C3 - P’ |
6 | Ligation control | None | None | ‘E - pSB1C3 - P’ |